RESUMO
The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28- T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166-CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9-based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.
Assuntos
Molécula de Adesão de Leucócito Ativado/metabolismo , Proliferação de Células/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Neoplasias/patologia , Receptores de Superfície Celular/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Molécula de Adesão de Leucócito Ativado/genética , Animais , Anticorpos Monoclonais/imunologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Técnicas de Inativação de Genes , Humanos , Células Jurkat , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Receptores Imunológicos , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
We have investigated the mechanism underlying the immunoregulatory function of membrane Ig-like transcript 3 (ILT3) and soluble ILT3Fc. microRNA (miRNA) expression profile identified genes that were downregulated in ILT3-induced human CD8(+) T suppressor cells (Ts) while upregulated in T cells primed in the absence of ILT3. We found that miR-21, miR-30b, and miR-155 target the 3'-untranslated region of genes whose expression was strongly increased in ILT3Fc-induced Ts, such as dual specificity phosphatase 10, B cell CLL/lymphoma 6, and suppressor of cytokine signaling 1, respectively. Transfection of miRNA mimics or inhibitors and site-specific mutagenesis of their 3'-untranslated region binding sites indicated that B cell CLL/lymphoma 6, dual specificity phosphatase 10, and suppressor of cytokine signaling 1 are direct targets of miR-30b, miR-21, and miR-155. Primed CD8(+) T cells transfected with miR-21&30b, miR-21&155, or miR-21&30b&155 inhibitors displayed suppressor activity when added to autologous CD3-triggered CD4 T cells. Luciferase reporter assays of miR-21 and miR-155 indicated that their transcription is highly dependent on AP-1. Analysis of activated T cells showed that ILT3Fc inhibited the translocation to the nucleus of the AP-1 subunits, FOSB and c-FOS, and the phosphorylation of ZAP70 and phospholipase C-γ 1. In conclusion, ILT3Fc inhibits T cell activation and induces the generation of Ts targeting multiple inflammatory miRNA pathways.
Assuntos
Linfócitos T CD8-Positivos/citologia , MicroRNAs/biossíntese , Receptores de Superfície Celular/fisiologia , Linfócitos T Reguladores/citologia , Regiões 3' não Traduzidas , Transporte Ativo do Núcleo Celular , Sítios de Ligação/genética , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Interferente Pequeno/farmacologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Imunológicos , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , TransfecçãoRESUMO
In extensive liver resection secondary to primary or metastatic liver tumors, or in living donor liver transplantation, strategies to quell deleterious inflammatory responses and facilitate regeneration are essential. The receptor for advanced glycation endproducts (RAGE) and myeloid differentiating factor 88 (Myd88) are implicated in the inflammatory response. To establish the contributions of RAGE vs. Myd88 signaling in extensive liver resection, we probed the effect of RAGE and/or Myd88, the latter primarily a key transducer of major toll-like receptors and also implicated in interleukin-1 (Il1) signaling, in a murine model of extensive (85%) hepatectomy. We report that, although Myd88 is thoroughly essential for survival via regulation of NF-κB and TNF-α, deletion of RAGE significantly improved survival compared to wild-type, Myd88-null, or RAGE-null/Myd88-null mice. RAGE opposes Myd88 signaling at multiple levels: by suppression of p65 levels, thereby reducing activation of NF-κB and consequent production of cyclin D1, and by suppression of Il6-mediated phosphorylation of Stat3, thereby down-regulating Pim1 and suppressing the hyperplastic response. Further, RAGE-dependent suppression of glyoxalase1, a detoxification pathway for pre-AGEs, enhances AGE levels and suppresses Il6 action. We conclude that blockade of RAGE may rescue liver remnants from the multiple signals that preclude adaptive proliferation triggered primarily by Myd88 signaling pathways.
Assuntos
Regeneração Hepática/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Receptores Imunológicos/fisiologia , Animais , Apoptose/fisiologia , Proliferação de Células , Produtos Finais de Glicação Avançada/metabolismo , Hepatectomia , Hepatócitos/citologia , Hepatócitos/metabolismo , Imunidade Inata , Regeneração Hepática/genética , Regeneração Hepática/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Regulação para CimaRESUMO
The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795-804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.
Assuntos
Injúria Renal Aguda/patologia , Quimiocina CXCL12/farmacologia , Nefropatias/patologia , Medula Renal/crescimento & desenvolvimento , Injúria Renal Aguda/fisiopatologia , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Indicadores e Reagentes , Nefropatias/fisiopatologia , Medula Renal/patologia , Medula Renal/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismoRESUMO
Ig-like transcript 3 (ILT3) is an inhibitory receptor expressed by tolerogenic dendritic cells. When human CD8(+) T cells are allostimulated in the presence of recombinant ILT3-Fc protein, they differentiate into antigenic specific T suppressor (Ts) cells that inhibit CD4 and CD8 T cell effector function both in vitro and in vivo. ILT3-Fc-induced CD8(+) Ts cells express high amounts of BCL6 that are crucial to their function. Knockdown of BCL6 from unprimed human T cells prevents their differentiation into Ts cells, whereas ex vivo overexpression of BCL6 converts CD8(+) T cells into Ts cells. NOD/SCID mice transplanted with human pancreatic islets and humanized by injection of human PBMCs tolerate the graft and develop BCL6(high) CD8(+) Ts cells when treated with ILT3-Fc before or after the onset of rejection. This indicates that ILT3-Fc acts through BCL6 and is a potent immunosuppressive agent for reversing the onset of allo- or possibly autoimmune attacks against pancreatic islets.
Assuntos
Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Receptores de Superfície Celular/imunologia , Tolerância ao Transplante/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Feminino , Técnicas de Silenciamento de Genes , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Imuno-Histoquímica , Transplante das Ilhotas Pancreáticas/imunologia , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-6 , Receptores Imunológicos , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
In this study, combined with field investigation and laboratory analyses, we assessed the distribution of soil organic carbon, nitrogen, phosphorous contents and their stoichiometric ratios, and the distribution of soil water stable aggregates along a soil texture gradient in the cropland of the Loess Plateau to understand the effect of soil texture and the regulation of soil aggregates on soil fertility in cropland. The results showed that, with the change from fine soils to coarse soils along the texture gradient (loam clayâ clay loamâ sandy loam), the contents of macroaggregates, organic carbon, nitrogen, phosphorous and their stoichiometric ratios decreased, while pH value and microaggregates content showed an opposite changing pattern. The contents of macroaggregates, organic carbon, nitrogen, phosphorous, and C/P and N/P were significantly increased, but pH value and microaggregates content were significantly decreased with increasing the soil clay content. Furthermore, the contents of organic carbon, nitrogen, phosphorous, and C/P and N/P increased with the increase of macroaggregates content. These results indicated that soil fertility in croplands at a regional scale was mainly determined by soil texture, and was regulated by soil macroaggregates.
Assuntos
Carbono , Nitrogênio , Fósforo , Solo , China , Produtos Agrícolas , ÁguaRESUMO
Lipoxin A(4) (LXA(4)) and aspirin-triggered 15-epi-LXA(4) are potent endogenous lipid mediators thought to define the inflammatory set-point. We used single prophylactic administrations of a synthetic aspirin-triggered lipoxin A(4) signal mimetic, ATLa, to probe dynamics of early host-donor interactions in a mouse model for the inflammation-associated multifactorial disease of allogeneic bone marrow transplant (BMT) -induced graft-vs.-host disease (GvHD). We first demonstrated that both host and donor are responsive to the ATLa signals. The simple and restricted regimen of a single prophylactic administration of ATLa [100 ng/mL to donor cells or 1 microg (approximately 50 microg/kg) i.v. to host] was sufficient to delay death. Clinical indicators of weight, skin lesions, diarrhea and eye inflammation were monitored. Histological analyses on day 45 post-BMT showed that the degree of cellular trafficking, particularly neutrophil infiltrate, and protection of end-organ target pathology are different, depending on whether the host or donor was treated with ATLa. Taken together, these results chart some ATLa protective effects on GvHD cellular dynamics over time and identify a previously unrecognized effect of host neutrophils in the early phase post-BMT as important determinants in the dynamics of GvHD onset and progression.-Devchand, P. R., Schmidt, B. A., Primo, V. C., Zhang, Q.-y., Arnaout, M. A., Serhan, C. N., Nikolic, B. A synthetic eicosanoid LX-mimetic unravels host-donor interactions in allogeneic BMT-induced GvHD to reveal an early protective role for host neutrophils.
Assuntos
Materiais Biomiméticos , Transplante de Medula Óssea , Eicosanoides , Reação Hospedeiro-Enxerto/fisiologia , Neutrófilos/fisiologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Eicosanoides/síntese química , Feminino , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/prevenção & controle , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Lipoxinas/administração & dosagem , Lipoxinas/síntese química , Lipoxinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Tolerogenic antigen presenting cells (APC) are characterized by high expression of the inhibitory receptors ILT3 and ILT4. We have engineered ILT3 and ILT4 cytoplasmic deletion mutants (ILT3delta and ILT4delta), which were transfected in the dendritic-like cell line KG1, to investigate ILT3 and ILT4's capacity to signal extracellularly. KG1.ILT3delta, similar to untruncated ILT3, inhibits T cell responses such as proliferation and cell-mediated cytotoxicity. In contrast, KG1.ILT4delta lost the suppressive activity of untruncated ILT4. This indicates that the inhibitory function of ILT4 relies entirely on the cytoplasmic region containing ITIM motifs. We further demonstrated that recombinant soluble ILT3 inhibits T helper and cytotoxic function while inducing the differentiation of CD8(+) Ts cells. Hence, Ts modulate APC function inducing inhibitory receptors, which in turn elicit the generation of Ts.
Assuntos
Imunossupressores/farmacologia , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunossupressores/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/farmacologia , Mutação/genética , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , TransfecçãoRESUMO
Bidirectional interactions between dendritic cells and Ag-experienced T cells initiate either a tolerogenic or immunogenic pathway. The outcome of these interactions is of crucial importance in malignancy, transplantation, and autoimmune diseases. Blockade of costimulation results in the induction of T helper cell anergy and subsequent differentiation of antigen-specific CD8+ T suppressor/regulatory cells (Ts). Ts, primed in the presence of inhibitory signals, exert their inhibitory function in an antigen-specific manner, a feature with tremendous clinical potential. In transplantation or autoimmunity, antigen-specific Ts can enforce tolerance to auto- or allo-antigens, while otherwise leaving the immune response to pathogens uninhibited. Alternatively, blockade of inhibitory receptors results in the generation of cytolytic CD8+ T cells, which is vital toward defense against tumors and viral diseases. Because CD8+ T cells are MHC Class I restricted, they are able to recognize HLA-bound antigenic peptides presented not only by APC but also on parenchymal cells, thus eliciting or suppressing auto- or allo-immune reactions.
RESUMO
To determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools.
Assuntos
Diferenciação Celular/genética , Medula Renal/metabolismo , Rim/metabolismo , Regeneração/genética , Proteínas de Transporte Vesicular/genética , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Rim/crescimento & desenvolvimento , Rim/patologia , Medula Renal/crescimento & desenvolvimento , Medula Renal/patologia , Camundongos , Células-Tronco/metabolismo , Tamoxifeno/farmacologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
OBJECTIVE: To study the role of T suppressor cells in immune tolerance to cardiac allografts in the rats. METHODS: Male DA rat hearts were transplanted to male Lewis rats using Ono's model and randomly divided into five groups: group 1: untreated, group 2: portal venous injection of 3 x 10(8) DA splenocytes to Lewis rat, group 3: intraperitoneal injection of cyclophosphamide (80 mg/kg) to Lewis rat, group 4: portal venous injection of 3 x 10(8) DA splenocytes combined with intraperitoneal injection of cyclophosphamide (80 mg/kg) to Lewis rat, 15 days later heart transplantation was performed. Group 5: intravenous injection 3 (108 splenocytes of group 4 to normal recipient, and then heart transplantation was performed. Mean survival time (MST), histological changes, mixed lymphocyte reaction (MLR) were measured after operation. RESULTS: The survival time of heart allografts in the group 4 [MST: (71.5 +/- 29.1) d, t = -14.063, -13.915, -13.777; P < 0.01] was significantly longer than in the groups of 1 [MST: (7.3 +/- 1.0) d], 2 [MST: (7.8 +/- 0.8) d], 3 [MST: (8.2 +/- 1.1) d ]. Only a few inflammatory cells infiltrated in cardiac allografts in the groups of 4 and 5. MLR in the groups of 4 and 5 were significantly decreased compared with those of normal control (t = 29.902, 23.047; P < 0.01). CONCLUSIONS: Portal venous injection of donor splenocytes combined with intraperitoneal injection of cyclophosphamide could induce immune tolerance to cardiac allografts. The immune tolerance could be transferred through splenocytes. T suppressor cells play an important role in the immune tolerance.
Assuntos
Facilitação Imunológica de Enxerto/métodos , Transplante de Coração/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/imunologia , Animais , Ciclofosfamida/uso terapêutico , Imunossupressores/uso terapêutico , Injeções Intraperitoneais , Transfusão de Linfócitos/métodos , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Transplante Homólogo/imunologiaRESUMO
OBJECTIVE: To study the methods and mechanisms of immune tolerance in cardiac transplantation. METHODS: Male DA rat hearts were transplanted to male Lewis rats using Ono's model and randomly divided into five groups: untreated, intravenous injection of 1 x 10(8) DA splenocytes to Lewis rat, intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat, intravenous injection of 1 x 10(8) DA splenocytes combined with intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat, multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide, 11 days later heart transplantation was performed. Mean survival time (MST), histological changes, mixed lymphocyte reaction (MLR), the role of interleukin-2 (IL-2) to MLR and the role of tolerant rat splenocytes to MLR were measured after operation. RESULTS: The survival time of heart allografts in the group of multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide [MST: (85.3 +/- 7.5) d, t = 0, P < 0.01] was significantly longer than in the groups of untreated [MST: (7.3 +/- 1.0) d], intravenous injection of 1 x 10(8) DA splenocytes to Lewis rat [MST: (7.9 +/- 0.9) d], intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat [MST: (8.1 +/- 1.2) d], intravenous injection of 1 x 10(8) DA splenocytes combined with intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat [MST: (25.8 +/- 3.5) d]. Only a few inflammatory cells infiltrated in cardiac allografts in the group of multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide. MLR in the group of multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide were significantly decreased compared with those of normal control (t = 0, P < 0.01). IL-2 could partly reversed the hyporesponsiveness of MLR in tolerant rats, the tolerance could be transferred in vitro. CONCLUSIONS: Multiple injection of donor splenocytes combined with intraperitoneal injection of cyclophosphamide to recipients could induce immune tolerance to cardiac allografts.
Assuntos
Transferência Adotiva , Transplante de Células , Ciclofosfamida/farmacologia , Facilitação Imunológica de Enxerto/métodos , Transplante de Coração/imunologia , Baço/citologia , Tolerância ao Transplante , Animais , Sobrevivência de Enxerto , Injeções Intravenosas , Isoantígenos/administração & dosagem , Isoantígenos/imunologia , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Ratos Endogâmicos , Ratos Wistar , Tolerância ao Transplante/efeitos dos fármacos , Transplante Heterólogo/imunologiaRESUMO
Psoriasis is a common chronic T-cell-mediated autoimmune skin disease, and traditional immunotherapies for psoriasis have focused on the direct inhibition of T cells, which often causes toxicity and lacks long-term effectiveness. Safe and effective therapeutic strategies are strongly needed for psoriasis. In this study, we show for the first time a significant accumulation of FLT3(+) CD11c(+) dendritic cells (DCs) in human psoriatic lesions and in the skin of experimental preclinical K14-VEGF transgenic homozygous mice, our animal model, although not an exact match for human psoriasis, displays many characteristics of inflammatory skin inflammation. SKLB4771, a potent and selective FLT3 inhibitor that we designed and synthesised, was used to treat cutaneous inflammation and psoriasis-like symptoms of disease in mice and almost completely cured the psoriasis-like disease without obvious toxicity. Mechanistic studies indicated that SKLB4771 treatment significantly decreased the number and activation of pDCs and mDCs in vitro and in vivo, and subsequent T-cell cascade reactions mediated by Th1/Th17 pathways. These findings show that targeted inhibition of FLT3, and hence direct interference with DCs, may be a novel therapeutic approach for the treatment of psoriasis.
Assuntos
Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Psoríase/imunologia , Tirosina Quinase 3 Semelhante a fms/imunologia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dermatite/imunologia , Modelos Animais de Doenças , Humanos , Queratina-14/genética , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Psoríase/tratamento farmacológico , Tiadiazóis/farmacologia , Tiadiazóis/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/genética , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
Allograft rejection remains a major limitation to successful solid organ transplantation. Here, we investigated the biosynthesis and bioactions of the pro-resolving mediators lipoxin A(4) and resolvin E1 in host responses to organ transplantation. In samples obtained during screening bronchoscopy after human lung transplantation, bronchoalveolar lavage fluid levels of lipoxin A(4) were increased in association with the severity of allograft rejection that was graded independently by clinical pathology. Lipoxin A(4) significantly inhibited calcineurin activation in human neutrophils, and lipoxin A(4) stable analogs prevented acute rejection of vascularized cardiac and renal allografts. Transgenic animals expressing human lipoxin A(4) receptors revealed important sites of action in host tissues for lipoxin A(4)'s protective effects. Resolvin E1 displays counter-regulatory actions for leukocytes, in part, via increased lipoxin A(4) biosynthesis, yet RvE1 administered (1µg, iv) to donor (days -1 and 0) and recipient mice (days -1, 0 and +4) was even more potent than a lipoxin stable analog (1µg, iv) in prolonging renal allograft survival (median survival time=74.0 days with RvE1 and 37.5 days with a LXA(4) analog). Together, these results highlight the potential for pro-resolving mediators in prolonging survival of solid organ transplants.
Assuntos
Ácido Eicosapentaenoico/análogos & derivados , Rejeição de Enxerto/fisiopatologia , Lipoxinas/biossíntese , Animais , Líquido da Lavagem Broncoalveolar/química , Ciclosporina/farmacologia , Ácido Eicosapentaenoico/biossíntese , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Coração , Humanos , Transplante de Rim , Transplante de Pulmão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ativação de Neutrófilo/efeitos dos fármacos , Receptores de Formil Peptídeo/biossíntese , Receptores de Formil Peptídeo/genéticaRESUMO
OBJECTIVE: The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action. RESEARCH DESIGN AND METHODS: NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L. RESULTS: Although human T-cell engraftment was similar in all groups, ILT3-Fc-treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks (P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8(+) T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc-treated mice and control recipients rejecting the grafts. CONCLUSIONS: The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Transplante das Ilhotas Pancreáticas/imunologia , Receptores de Superfície Celular/imunologia , Linfócitos T/imunologia , Transplante Heterólogo/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/genética , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Experimental/imunologia , Feminino , Rejeição de Enxerto/prevenção & controle , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/imunologia , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase , RNA/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/uso terapêutico , Receptores Imunológicos , Ensaio de Cápsula Sub-Renal , Transplante Homólogo/imunologiaRESUMO
Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.
Assuntos
Adenocarcinoma/imunologia , Neoplasias Colorretais/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Melanoma/imunologia , Neoplasias Pancreáticas/imunologia , Receptores de Superfície Celular/fisiologia , Linfócitos T Reguladores/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Processamento Alternativo , Animais , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Anergia Clonal , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Rejeição de Enxerto/patologia , Humanos , Melanoma/metabolismo , Melanoma/patologia , Glicoproteínas de Membrana , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/genética , Receptores Imunológicos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/patologia , Evasão TumoralRESUMO
To observe whether bone marrow stromal cell line QXMSC1 (H-2(d)) engineered to secrete IL-3 and IL-6 can improved the hematopoiesis in allogeneic bone marrow transplantation (allo-BMT) mice, the stromal cell line QXMSC1IL-3/IL-6 was established by QXMSC1 cells transduced with the recombined retrovirus vector pL3SN containing mouse IL-3 cDNA and pL6SN containing human IL-6 cDNA. The lethally irradiated C57BL/6 (H-2(b)) mice were engrafted with bone marrow cells (1 x 10(7) cells/mouse BALB/c mice, H-2(d)) in which T cells were depleted by anti-Thy1.2 monoclonal antibody adding complement, and QXMSC1IL-3/IL-6 cells (5 x 10(5)/mouse) were co-infused at same time. The hematopoiesis of recipient mice was observed in 20 and 40 days post-transplantation. Blood RBC and WBC were counted, and nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM were assayed in recipient bone marrow. Results showed that IL-3 and IL-6 were stably expressed in QXMSCQIL-3/IL-6 cells. Compared with BMT and co-infusion with QXMSC1 or QXMSC1 pLXSN cell groups, co-graft with QXMSC1IL-3/IL-6 cells increased the number of blood RBC and WBC in the recipients, and also significantly increased nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM in recipient bone marrow. It is concluded that the marrow stromal cells transduced with IL-3/IL-6 cDNA improve the hematopoiesis in allo-BMT mice. Co-graft with QXMSC1IL-3/IL-6 cells has synergistic effect in accelerating hematopoietic reconstitution.
Assuntos
Transplante de Medula Óssea/métodos , Terapia Genética , Hematopoese , Interleucina-3/genética , Interleucina-6/genética , Animais , Células da Medula Óssea/fisiologia , Feminino , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Estromais/fisiologia , Células Estromais/transplante , Transdução GenéticaRESUMO
AIM: To construct CTLA4Ig adenovirus vectors (AdCTLA4Ig) by homologous recombination and study their activity, and to employ the vectors to induce cardiac transplantation tolerance by gene therapy. METHODS: CTLA4Ig gene was cloned to pCA13 adenovirus shuttle plasmid by recombination strategy. Construction of CTLA4Ig adenovirus vectors was performed by homologous recombination of pCA13 plasmid containing CTLA4Ig gene with adenovirus helper plasmid, followed by packaged with 293 cells. Expression and secretion of CTLA4Ig was confirmed by RT-PCR, SDS-PAGE and Western blot. The inhibitory effect of supernate of 293 cells infected with AdCTLA4Ig on MLR in-vitro was observed. A biological activity of CTLA4Ig adenovirus vectors was determined by AdCTLA4Ig gene therapy in rats in-vivo. RESULTS: The Construction of CTLA4Ig adenovirus vector was successful. It was confirmed that the supernatant of 293 cells infected with AdCTLA4Ig could inhibit MLR in-vitro. It was also showed that CTLA4Ig adenovirus vectors could induce transplantation tolerance and prolong allograft survival when they were administrated in rats in-vivo. CONCLUSION: CTLA4Ig adenovirus vectors successfully constructed can infect 293 package cells and secrete CTLA4Ig. The CTLA4Ig protein can inhibit T cell activation. The CTLA4Ig adenovirus vectors can be employed to gene therapy in-vivo, and induce transplantation tolerance.